EP2302037A1 - Verfahren zur nichtautologen Knorpelregeneration - Google Patents

Verfahren zur nichtautologen Knorpelregeneration Download PDF

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EP2302037A1
EP2302037A1 EP09010305A EP09010305A EP2302037A1 EP 2302037 A1 EP2302037 A1 EP 2302037A1 EP 09010305 A EP09010305 A EP 09010305A EP 09010305 A EP09010305 A EP 09010305A EP 2302037 A1 EP2302037 A1 EP 2302037A1
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Prior art keywords
mcdc
film
cartilage
mammal
chondrocyte
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English (en)
French (fr)
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Gila Maor
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CARTICURE Ltd
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CARTICURE Ltd
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Priority to EP10183141A priority Critical patent/EP2264149A1/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates in general to the field of tissue engineering, and in particular to methods for repair and regeneration of diseased and injured bone and cartilage tissue by allotransplantation or xenotransplantation of perinatal mandibular condyle-derived chondrocytes.
  • Tissue engineering may be defined as the art of structurally and functionally reconstructing mammalian tissues (Hunziker, 2002). In general, tissue engineering provides cells per se or a scaffold that serves as a support onto which cells may attach, proliferate, and synthesize tissue in situ, to restore tissue lost due to disease, trauma or the aging process.
  • Articular joints are a vital component of the musculoskeletal system. Freely moving joints (ankle, elbow, hip, knee, shoulder, and those of the fingers, toes and wrist) are known as diarthrodial or synovial joints and are critical for body movement. Synovial joints have several distinguishing characteristics, including articular cartilage, a hyaline cartilage covering the ends of the opposing bones and a joint cavity, a space that is filled with lubricating synovial fluid. Articular cartilage is structurally organized and consists of specialized cartilage cells termed "chondrocytes" embedded in an intercellular "cartilage matrix", which is rich in proteoglycans, collagen fibrils (primarily type II), other proteins, and water.
  • synovial fluid In animal joints, the synovial fluid, articular cartilage, and the supporting bone form the weight-bearing system of the body. While synovial joints are subject to an enormous range of loading conditions on a daily basis, the cartilage surfaces undergo little wear and tear under normal circumstances. Breakdown of the joint cartilage as a result of disease, senescence or wear leads to arthritis.
  • Cartilage and bone diseases include highly debilitating diseases such as osteoarthritis, articular cartilage injury, meniscal disorders and joint infections for which no optimal therapies are currently available. Repair of arthritic joints requires orthopedic surgery to regenerate the degenerated cartilage by a prosthesis or by a biological graft.
  • cartilage or bone tissue is treated by administering chondrocytes isolated from autogeneic articular or auricular cartilage.
  • Autologous chondrocyte implantation, or ACI is a procedure wherein cartilage cells are isolated from a subject's knee, cultured as discrete cells or as aggregates, and subsequently implanted into the subject per se or in combination with a scaffold or matrix.
  • the inventor of the present invention has developed a model for the redifferentiation of chondrocytes, which were isolated from 3-day old mouse mandibular condylar tissue (Reiter et al, 2002).
  • US Patent Application Publication No. 2004/0175826 of the inventor of the present invention, teaches a method of generating cultured chondrocytes from mandibular condyle tissue isolated from 3 day old mice. That application further discloses treating a subject by administering syngeneic or allogeneic mandibular condyle-derived chondrocytes to a subject in need thereof.
  • cultured cells from a genetically engineered animal such as a pig were suggested.
  • Cartilage has been considered an immuno-privileged tissue. Allograft cartilage transplantations have been successfully performed both in experimental models of damaged articular cartilage in animals (Lu, et al, 2005; Moskalewski et al, 2002) and in patients suffering from articular lesions. Moreover, it has been shown that a cartilage capsule may serve as an immunoisolation barrier of transplanted insulin producing-Langerhans cells, utilizing the immunoprivileged properties of the chondrocyte matrix (Pollok, et al., 2001a; Pollok et al, 2001b).
  • cartilage is an avascular tissue that is also devoid of lymph vessels.
  • cartilage being an immunoprivileged tissue is mainly due to the fact that most of its cellular antigenic sites are hidden within dense extracellular matrix.
  • Elves (1976) stated that cartilage graft is therefore antigenic but only feebly immunogenic, as the matrix proteoglycans protect the cells from the afferent arm of the immune response.
  • compositions for xenotransplantation for human chondral defects are known, but none have proven to be effective.
  • Fuentes-Boquete et al used porcine chondrocytes to repair human cartilage lesions. Following 8-12 weeks, repair tissue filled near 30-40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline-like region. The repaired tissue showed positive immunostaining for both type I and II collagen, whereby type I collagen is a marker for the presence of fibrocartilage.
  • Soft tissue xenografts for cartilage repair is disclosed in US Patent No. 6,758,865 . That tissue is prepared by treating the graft with a glycosidase, followed by sialylation treatment, in order to prepare substantially non-immunogenic xenografts for soft tissue repair.
  • US Patent No. 6,645,764 discloses human neocartilage, having multiple layers of cells surrounded by a substantially continuous insoluble glycosaminoglycan and collagen-enriched hyaline extracellular matrix.
  • the neocartilage serves as replacement tissue for diseased or injured cartilage.
  • That invention is exemplified by allogeneic neocartilage, although according to that disclosure the neocartilage may comprise avian or mammalian chondrocytes derived from transgenic animals, which have been genetically engineered to prevent immune-mediated xenograft rejection.
  • the neocartilage composition is a substantially continuous layer of tissue at least two cell layers thick. After 14 days of growth, the neocartilage was between 10 and 15 cell layers thick. The neocartilage can be grown to various size specifications to facilitate implantation.
  • US Patent Application Publication 2004/0082063 teaches three-dimensional macromass cultures of, inter alia , chondrocytes. The tissue-like masses are disclosed to be useful for tissue replacement.
  • the present invention provides methods for the treatment of cartilage and bone disorders comprising mandibular condyle-derived chondrocytes (MCDC) isolated from perinatal mammals for the preparation of a xenograft or allograft composition.
  • MCDC mandibular condyle-derived chondrocytes
  • both the allograft and xenograft are substantially non-immunogenic and may be administered in the absence of immunosuppressive therapy.
  • the cells are versatile and can be utilized per se , in combination with a scaffold or can be prepared as a chondrocyte sheet, also designated hereinbelow as film.
  • the present inventor discloses for the first time that a flexible chondrocyte film comprising porcine perinatal MCDC repairs large cartilage defects in a xenogeneic goat model.
  • murine and porcine mandibular condyle-derived chondrocytes (MCDC) isolated from perinatal and neonatal animals do not evoke a host immune response when transplanted into damaged knees of adjuvant induced arthritic (AIA) rats.
  • AIA adjuvant induced arthritic
  • mouse and porcine-derived MCDC's replenished the articular lesions of rats with no sign of white blood cell infiltration.
  • the cells and the chondrocyte film when transplanted into a cartilage lesion, develop into durable, functional articular hyaline cartilage with no signs of fibrillar matrix organization, the structural feature typifying fibrocartilage. Fibrocartilage has poor mechanical properties and degenerates over time. Importantly, the cells adapt themselves to the original anatomic shape of the articular cartilage and do not hyperproliferate in vivo, thereby reducing the risk of a tissue overgrowth in the joint.
  • the present invention provides a flexible chondrocyte film for the treatment of an orthopedic disorder, the chondrocyte film comprising from one to about ten layers of mammalian perinatal mandibular condyle derived chondrocytes (MCDC), wherein the MCDC are surrounded by an insoluble matrix, wherein the insoluble matrix comprises type II collagen.
  • MCDC mammalian perinatal mandibular condyle derived chondrocytes
  • the film comprises about 2 to about 5 layers of MCDC, preferably from about 2 to about 3 cell layers of MCDC.
  • the cartilage film retains its capacity to undergo de novo proliferation and differentiation in vivo.
  • the chondrocyte film is capable of regenerating hyaline cartilage in vivo.
  • the regenerated hyaline cartilage is substantially free of fibrocartilage.
  • the matrix further comprises at least one proteoglycan. In preferred embodiments the matrix comprises aggrecan.
  • the MCDC are isolated from a perinatal donor mammal, wherein the donor of the MCDC are allogeneic or xenogeneic to the recipient subject.
  • the mammal is a neonate and the neonatal age is less than 7 days old. In some embodiments the neonate is about 3 days old, or preferably about 24 hours old. In other embodiments the mammal is a fetus.
  • Suitable mammals for the isolation of MCDC include a human and non-human mammal. Suitable mammals include, inter alia rat, mouse, rabbit, guinea pig, goat, lamb, sheep, calf, cow, dog and pig. In one preferred embodiment the mammal is a human.
  • compositions of the present invention are substantially non-immunogenic , namely do not elicit an immune response that is detrimental to the retention of the graft.
  • the composition is administered to a subject in the absence of an immunosuppressive therapy.
  • the present invention provides a method for the treatment of an orthopedic disorder in a subject in need thereof, the method comprising the step of administering to a site requiring chondrocytes in the subject a flexible chondrocyte film, the chondrocyte film comprising from one to about ten layers of mammalian mandibular condyle derived chondrocytes (MCDC), wherein the MCDC are surrounded by an insoluble matrix, wherein the insoluble matrix comprises type II collagen and aggrecan.
  • MCDC mammalian mandibular condyle derived chondrocytes
  • the MCDC are isolated from a perinatal donor mammal, wherein the donor of the MCDC are allogeneic or xenogeneic to the recipient subject.
  • the mammal is a neonate and the neonatal age is less than 7 days old.
  • the neonate is less than about 7 days old. In some embodiments the neonate is less than about 3 days old, or preferably less than about 24 hours old. In other embodiments the mammal is a fetus.
  • Suitable mammals for the isolation of MCDC include human and non-human mammals. Suitable mammals include, inter alia rat, mouse, goat, lamb, calf, dog and pig. In other embodiments the mammal is a human.
  • the MCDC are allogeneic to the recipient subject. In other embodiments the MCDC are xenogeneic to the recipient subject.
  • the subject is a human subject and the MCDC are allogeneic. In other embodiments the subject is human and the MCDC are xenogeneic. In some embodiments the xenogeneic MCDC derive from a mammal selected from a dog and a pig.
  • compositions of the present invention are substantially non-immunogenic.
  • composition is administered to a subject in the absence of an immunosuppressive therapy.
  • the orthopedic disorder is a lesion in the cartilage resulting from disease or injury.
  • the disease is a cartilage degenerative disease selected from osteoarthritis and rheumatoid arthritis.
  • the lesion results from trauma, for example, from a sports injury or accident.
  • the orthopedic disorder is a bone disorder, including for example a bone fracture or lesion.
  • the injury or diseased tissue includes cartilage and subchondral bone.
  • the chondrocyte film is topically administered to the site of injury or disease, via surgical techniques including open knee surgery and arthroscopy.
  • the present invention provides a method for preparing a flexible chondrocyte film comprising about one to about ten layers of mammalian perinatal MCDC, wherein the chondrocytes are surrounded by an insoluble matrix, the matrix comprising type II collagen and aggrecan, the method comprising the steps of:
  • the present invention further provides the use of mammalian perinatal MCDC for the preparation of a flexible chondrocyte film useful in the treatment of an orthopedic disorder.
  • the chondrocyte film comprises about one to about ten layers of MCDC, wherein the chondrocytes are surrounded by an insoluble matrix, the matrix comprising type II collagen.
  • the matrix further comprises a proteoglycan. In various preferred embodiments the matrix comprises aggrecan.
  • the present invention further provides the use of a flexible chondrocyte film comprising about one to about ten layers of mammalian perinatal MCDC, wherein the chondrocytes are surrounded by an insoluble matrix, the matrix comprising type II collagen and aggrecan for treating an orthopedic disorder.
  • the present invention provides a method for the treatment of an orthopedic disorder in a subject in need thereof, the method comprising the step of administering to the subject a composition comprising a therapeutically effective amount of mandibular condyle derived chondrocytes (MCDC) isolated from a perinatal donor mammal, wherein the perinatal donor is allogeneic or xenogeneic to the subject.
  • MCDC mandibular condyle derived chondrocytes
  • the mammal is a neonate and the neonatal age is less than 3 days old. In preferred embodiments the neonate is about 24 hours old.
  • the cells are transplanted per se.
  • the composition of the present invention does not require the use of a scaffold but in certain embodiments the cells are transplanted together with a scaffold, for example a biocompatible scaffold.
  • the present invention provides the use of a therapeutically effective amount of perinatal mandibular condyle derived chondrocytes (MCDC) isolated from an allogeneic or xenogeneic perinatal mammal for the preparation of a composition useful in the treatment of an orthopedic disorder in a subject in need thereof.
  • MCDC mandibular condyle derived chondrocytes isolated from an allogeneic or xenogeneic perinatal mammal
  • the composition comprises fetal or neonatal MCDC.
  • the present invention provides methods of using allogeneic or xenogeneic perinatal mandibular condyle derived chondrocytes for cartilage and bone regeneration and repair.
  • the MCDC have a number of characteristics, which render them advantageous in clinical applications.
  • the advantageous properties of the matrices of the invention include:
  • cartilage refers to a specialized type of connective tissue that contains chondrocytes embedded in an extracellular matrix.
  • types of cartilage have been identified which differ in their histological and mechano/physical properties and include, hyaline cartilage, fibrous cartilage, and elastic cartilage.
  • Articular cartilage that covers the apical regions of bones at the joints is composed of hyaline cartilage.
  • the biochemical composition of cartilage differs according to type but characteristically comprises fibers: collagens and/or elastic fibers, ground-amorphous substance comprised mainly of glycosaminoglycans proteoglycans, other proteins and water.
  • chondrocytes refers to cells, which are capable of producing components of cartilage tissue.
  • Hyaline cartilage the most abundant form of cartilage, is glass smooth, glistening and bluish white in appearance, although older or diseased tissue tends to lose this appearance.
  • Articular cartilage is characterized by specialized cartilage cells termed “chondrocytes” embedded in a “cartilage matrix", which is rich in cartilage specific highly sulfated proteoglycans-denoted aggrecans, type II collagen fibrils along with other minor types, e.g., types IX and XI, other proteins, and water.
  • cartilage tissue is neither innervated nor penetrated by the vascular or lymphatic systems, in the mature joint of adults, the underlying subchondral bone tissue is innervated and vascularized. Beneath this bone plate, the bone tissue forms trabeculae, containing the marrow. In immature joints, articular cartilage is underlined by only primary bone trabeculae. A portion of the meniscal tissue in the knee joint (referred to as the "interarticular" cartilage) consists mainly of fibrous cartilage.
  • Neocartilage refers to cartilage, which is produced ex vivo , and can be implanted per se to replace cartilage.
  • Fibrous cartilage is characterized by the presence of type I cartilage.
  • the presence of type I collagen in repaired cartilage is believed to be unfavorable for tissue repair (Briggs, et al., 2003).
  • the term "substantially free of fibrous cartilage” refers to regenerated cartilage tissue that has less than about 20% fibrous cartilage, preferably less than about 10%, preferably less than about 5% fibrous cartilage as measured by the presence of type I collagen.
  • Cartilage film and “chondrocyte film” are used herein interchangeably and refer to a flexible matrix comprising chondrocytes embedded within an insoluble matrix, the matrix being about one to about ten cell layers thick. Thee insoluble matrix is secreted by the chondrocytes.
  • a flexible film refers to the capability of being folded, shaped or bent while maintaining integrity.
  • a flexible film is a pliable film.
  • a flexible chondrocyte film can be molded or bent to fit the contours of the tissue in need of regeneration.
  • mandibular condyle derived chondrocytes refers to chondrocytes isolated from the mandibular condyle of a neonate.
  • the mandibular condyle refers to the growth center in the lower jawbone, where the jaw articulates with the temporal bone of the skull in a vertebrate.
  • the mandibular condyle is a unique source of cartilage since it serves two distinct developmental objectives: during the early postnatal period it serves as the growth center of the mandible following normal cascade of endochondral ossification, and upon growth cessation it turns into articular cartilage.
  • the MCDC cells are able to develop into fully functional hyaline cartilage in vitro and in situ and express the molecular markers associated therewith; functionally and structurally repair or replace damaged or diseased cartilage tissue; exhibit a high proliferation rate in vitro yet do not over proliferate in vivo, thus obviating the risk of tissue repair with fibrocartilage; can be transferred from one substratum to another while renewing their proliferative and chondrogenic features, thus producing new hyaline cartilage in the new substratum.
  • repair and “regeneration” refer to the proliferation, growth, development and or restoration of tissue in situ that has been damaged by trauma, disease or injury.
  • non-autologous refers to tissue or cells which originate from a donor other than the recipient.
  • Non-autologous can refer to for example allogeneic or xenogeneic.
  • autogeneic and autologous as in autogeneic graft, autologous chondrocytes etc, refers to a graft, cartilage, etc., in which the donor and recipient are the same individual.
  • allogeneic refers to a donor and a recipient of the same species;
  • seyngeneic refers to a donor and recipient with identical genetic make-up (e.g. identical twins or autogeneic) and
  • xenogeneic refers to donor and recipient of different species.
  • substantially non-immunogenic or “substantially non-antigenic” means that the graft, implant or transplant does not elicit an immune response that is detrimental to the retention of the graft.
  • a “subject” refers to a recipient or host of the composition of the present invention. In some embodiments the subject is a human subject.
  • transplantation or “implantation” refers to the insertion of the film or the injection, infusion or insertion of the composition of the invention into a subject, whereby the composition serves to replace, fully or partially, tissue that has been damaged, diseased or removed.
  • Xenotransplantation refers to the transplantation of cells, tissues or organs from one species to another such as from mice to rats or pigs to humans. Such cells, tissues or organs are called xenografts (xenotransplants).
  • a graft refers to transplanted or implanted cells or tissue.
  • a graft as used herein refers to transplanted chondrocyte film or transplanted MCDC.
  • perinatal refers to a period from before birth to shortly after birth. In a human, perinatal refers to the period beginning with completion of the twentieth week of gestation and ending about 28 days after birth.
  • neonatal refers to a period beginning at birth and ending at about 28 days after birth.
  • fetal refers the period beginning at about 20 weeks post gestational until birth.
  • biocompatible refers to a material which has low toxicity, acceptable foreign body reactions in the living body, and affinity with living tissues.
  • cell-bearing refers to the capacity of a material to support cells.
  • scaffold refers to a solid or semi-solid material, which can be used as a delivery vehicle for cells and bioactive agents.
  • Biocompatible refers to low toxicity, clinically acceptable levels of foreign body reactions in the living body, and affinity with living tissues.
  • proteoglycan refers to a special class of proteins that are heavily glycosylated.
  • a proteoglycan is made up of a core protein with numerous covalently attached high sulphated glycosaminoglycan chain(s).
  • Non-limiting example of extracellular matrix proteoglycans include aggrecan and certain collagens, such as collagen IX. The presence of aggrecan in a chondrocyte culture is a marker of articular hyaline cartilage.
  • Type I collagen is generally a component of fibroblast forming tissue, scar tissue, tendons and the organic part of bone.
  • the expression of type I collagen is unfavorable to the development of articular hyaline cartilage, while the expression of type II collagen is favorable.
  • glycosaminoglycan refers to a long unbranched polysaccharide molecules found on the cell surface or within the extracellular matrix.
  • glycosaminoglycan include heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, hyaluronic acid, hexuronyl hexosaminoglycan sulfate, and inositol hexasulfate.
  • Xenografts and allografts offer tremendous advantage over autologous transplant.
  • Use of xenogeneic cultures, if immunologically neutral, allows maintenance of a small number of cell cultures for virtually all recipient subjects. This reduces patient trauma from tissue harvest, reduces the expense of generating primary cultures, reduces the waiting time required for a surgical procedure and obviates the risk that the autologous chondrocyte culture will not, for some reason, proliferate.
  • the above risks are further minimized with regard to the MC derived chondrocytes of the present invention.
  • the MCDC are isolated from xenogeneic or allogeneic perinatal mammals and shown to be substantially non-immunogenic and non-antigenic, thereby obviating the need for high doses of immunosuppressive drugs. Without wishing to be bound to theory, these features result from the rapid differentiation rate and accompanying high levels of extracellular matrix, which envelops the cells.
  • the cultured cells of the present invention can be administered in-vivo, and in particular in situ , for the repair and or replacement of lost or diseased skeletal tissue.
  • Skeletal tissue refers to hard tissues such as bone and subchondral bone and to articular or meniscal cartilages.
  • the methods of the present invention are suitable for human and veterinary applications.
  • the recipient of the MCDC is a human or non-human mammal selected from horse, dog, goat or sheep. In certain embodiments the recipient is a human subject.
  • the method of treatment comprises administering a therapeutically effective dose of allogeneic or xenogeneic MCDC to a subject in need thereof.
  • therapeutically effective dose refers to an amount sufficient to effect a beneficial or desired clinical result, wherein the clinical result is repair or regeneration of cartilage or bone tissue.
  • cartilage tissue is repaired.
  • treating refers to alleviating, attenuating, palliating or eliminating the symptoms of a disease, slowing, reversing or arresting the progression of the disease, or curing the disease.
  • the disease refers to any medical disease, disorder, condition, or syndrome, or to any undesired and/or abnormal physiological, morphological, and/or physical state or condition.
  • the disease is a chondral disease
  • the disease is an osteochondral disease.
  • the present invention provides a method for treating orthopedic defects inter alia articular cartilage lesions arising from trauma such as an accident or sports injury or disease such as osteoarthritis. The method is preferably applied to treat the disease in a mammalian subject, preferably a human subject.
  • Osteoarthritis usually affects only one or two major joints, and often affects the knee.
  • the etiology of knee osteoarthritis is not known; it is thought to be simply a process of "wear and tear". Some conditions may predispose the knee to osteoarthritis, for example, a previous fracture that involved the joint, or by lesser injuries that may have torn ligaments or menisci.
  • Abnormal development of the bones, such as bow legs may cause the knee to wear out sooner than normal.
  • the cartilage cushion is either thinner than normal (leaving bare spots on the bone), or completely absent resulting in bare bones grinding against each other, and often resulting in damage to the subchondral bone.
  • fragments of cartilage floating in the joint may cause inflammation in the joint lining. X-rays show the "joint space" to be narrowed and irregular in outline.
  • RA rheumatoid arthritis
  • Osteonecrosis, or spontaneous osteonecrosis of the knee is a rare condition, which may cause knee pain.
  • compositions of the present invention are useful in the repair of bone defects including breaks and gaps.
  • the present invention provides a chondrocyte film comprising from one to about 10 layers of MCDC, wherein the chondrocytes are surrounded by an insoluble matrix, wherein the insoluble matrix comprises type II collagen and aggrecan.
  • the film comprises about 2 to about 5 layers of MCDC, preferably from about 2 to about 3 cell layers.
  • the cartilage film can undergo proliferation and differentiation in situ .
  • the chondrocyte film can be implanted into a host, for example to repair a cartilage defect in the knee.
  • the film can be implanted during open knee surgery or in the less invasive surgical procedure known as arthroscopy.
  • the chondrocyte film may need to be affixed to its target tissue, for example, by suturing or application of surgical glue.
  • the chondrocyte film is allogeneic or xenogeneic to the recipient.
  • allogeneic or xenogeneic MCDC are transplanted to a subject.
  • the therapeutic composition according to the present method is useful for inducing regeneration or repair of cartilage and bone lesions.
  • the composition can also include one or more pharmaceutically acceptable excipients.
  • a "pharmaceutically acceptable excipient” refers to any substance suitable for maintaining and delivering the cells of the composition to an in vivo site (i.e., a cartilage lesion).
  • Preferred pharmaceutically acceptable excipients are capable of maintaining the viability of the cells.
  • pharmaceutically acceptable excipients include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, and the like.
  • Aqueous carriers can contain suitable additives required to approximate the physiological conditions of the recipient, for example, by enhancing stability and isotonicity. Suitable additives include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Additives can also include preservatives.
  • the MCDC can be administered as isolated, cultured cells to a subject in situ , i.e. directly to the lesion.
  • the cells may be associated with a biocompatible scaffold.
  • Suitable scaffolds include those made of synthetic or natural materials. Natural materials include cross-linked or non-crosslinked hyaluronic acid, collagen, chitosan, alginate, fibrin, peptide hydrogels, demineralized bone and the like. Synthetic materials include polyethylene glycols, poly(DL-lactic-co-glycolic acid) (PLGA), polyurethane foam, and the like.
  • Neonatal piglet 24 hr old, male or female, ⁇ 1.6 kg weight was deeply anesthetized with 5 ml Iustil (200mg/ml sodium pentobarbiton, injected into the heart).
  • Mandibular condyles were aseptically dissected, freed of any soft tissue and subjected to successive collagenase digestion (37°C; 0.1 % type II collagenase, cat No. C-6885, Sigma Co., St. Louis, MO, USA). Following the 1 st digestion (25 min), which mainly separates adjacent soft tissues, the next 4 successive digestions (45 min each) yielded homogenous population of chondrocytes. These mandibular condyle derived chondrocytes are denoted MC. For the sake of comparison, chondrocytes were also separated from the femoral distal condyles and are referred to herein AC.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • penicillin/streptomycin cat No. 030421, 030201, 030311, respectively, Biological Industries, Bet Ha'Emek, Israel
  • 100 ⁇ g/ml ascorbic acid and 10 mM ⁇ -glycerophosphate cat. No. A-4544, C-6251, respectively, Sigma Co., St. Louis, MO, USA.
  • Cells are plated at a concentration of 5 ⁇ 10 5 cells/ml in 35 mm Dia culture dishes at 37°C, 5% CO 2 and maximal humidity. Medium was changed every 48 hr. Every 3-4 days the cells were split. For the present experiments, cells from the second passage (P2) were used.
  • MTT assay Cell viability was measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, cat No. M2128, Sigma , St. Louis, MO, USA) assay. The assay is based on the capability of mitochondrial dehydrogenase in intact cells to oxidize MTT into the blue/brownish insoluble formazan.
  • Chondrocytes were incubated with 20 ⁇ Ci/ml of [ 35 S]-Na 2 SO 4 for 6 hr at 37°C or 10 ⁇ Ci/ml of [ 35 S]-Na 2 SO 4 for 16 hr at 37°C.
  • cells were washed twice with PBS (wash fluid is kept and combined with further radioactivity extracts), the medium and cell layer were digested separately with papain (250 ⁇ g/ml) for 2 h at 60°C in the presence of 100 ⁇ g of chondroitin sulphate-A (cat. No. C9819, Sigma), which serves as a carrier. Chondrocytes were then extracted in 4 M guanidine-HCl buffer / 50 mM sodium acetate buffered at pH 7.2, in the presence of protease inhibitors.
  • CPC cetylpyridinium chloride
  • the precipitate was collected by centrifugation and washed with 1ml of 1% CPC, this step was repeated twice. The final precipitate was dissolved in formic acid and radioactivity was counted.
  • Cell lysate was prepared in ice cold RIPA buffer supplemented with protease inhibitors: 10 ⁇ g/ml PMSF, 5 ⁇ g/ml Aprotonin, 5 ⁇ g/ml Trypsin inhibitor and 10 ⁇ g/ml Benzamidine. Lysates proteins were separated in reducing sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose membrane. Blots were incubated with one of the following: mouse anti-collagen type II (cat. No. MAB 887 Chemicon International, Inc, Temecula, CA, USA), mouse anti-Proliferating Cell Nuclear Antigen (PCNA) (cat No.
  • PCNA Mouse anti-Proliferating Cell Nuclear Antigen
  • ISH In situ Hybridization
  • Probe labeling Digoxygenin (DIG)-labeled anti sense RNA probes were prepared in a two steps polymerase chain reaction (PCR) method: amplification of specific gene insert, amplification of the gene insert conjugated to promoters at each side. The following probes were used for ISH:
  • the second primer pair has T3 and T7 promoter sequences at the ends.
  • Antisense mRNA was labeled using 1 ⁇ g of gel-purified PCR product (QIAquick® gel extraction kit, Qiagen) and DIG RNA labeling kit (Boehringer Mannheim, Germany), following the manufacturer's instructions.
  • Hybridization MCDC cells cultured on cover slips were pre-treated with cold methanol to increase membrane penetration followed by quenching endogenous peroxidase with 3% H 2 O 2 in methanol. Cells were then treated with12.5 ⁇ g/ml proteinase K for 15 min, (stopped with 2 mg/ml glycine), and acetylated with 0.5% acetic anhydride in 0.1 M Tris at pH 8.0; sections are post-fixed with 4% paraformaldehyde/phosphate buffered saline (PBS).
  • PBS paraformaldehyde/phosphate buffered saline
  • Prehybridization for 10' in 2 x standard saline citrate (SSC) is followed by one hr in hybridization buffer: 50% formamide 0.5 mg/ml salmon sperm DNA, 4xSSC, 1x Denhardt's, 200 U/ml heparin, 5% dextran sulfate and 0.01% sodium dodecyl sulfate (SDS).
  • Hybridization was performed overnight (18hr) at 42°C and maximal humidity with 5ng/ ⁇ l digoxigenin labeled antisense RNA probe (see above). For all probes used, digoxigenin labeled sense RNA probes served as negative controls.
  • necropsies of osteochondral are harvested from the treated lesions, the untreated lesions and from parallel healthy tissues in the counter (intact) knee.
  • Specimens are immersed in Phosphate-Buffered Saline (PBS; 2.67 mM KCl, 1.47 mM KH 2 PO 4 , 138 mM NaCl, 8.1 mM Na 2 HPO4 ⁇ 7H 2 O at a pH 7.2) and protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 2 mM disodium ethylenediamine tetraacetate, 5 mM benzamidine-HCl, and 10 mM N-ethylmaleimide). Tissues are assayed within 5 hrs of their harvesting.
  • Stiffness of the tissue is assessed by indentation test using a universal testing apparatus 4502'-Instron-BioplusTM, and a 0.3mm plane ended indenter.
  • the loading protocol consists of application of a "tare displacement” (for 30 s) and then four "test displacements" of 3 seconds (s) duration each. For each tare displacement-load at 0 s and 3 s is noted. Tare displacement is 25 micrometer, test displacements are 50,100, 200, and 300 Micrometers.
  • Indentation Stiffness is computer calculated as the slope of load-displacement data, in units of N/mm (with no need to regard indenter diameter).
  • Mandibular condyles are ascetically removed from 20-week-old fetuses of deliberate abortions, explants are immediately transferred into cold Hank's buffer. Cartilaginous zone is removed by cutting at the mineralization front of the condyle using dissecting microscope. Cartilage tissue is subjected to gradually enzymatic digestions (0.1 % collagenase type II), as described for porcine-derived MCDC. Separated chondrocytes are cultured under the same conditions as porcine MCDC.
  • the chondrocyte yield from mandibular condyle is higher than that from knee articular cartilage.
  • Morphological appearance of the cultures was followed by phase microscopy. Results presented in figure 1 clearly show that the mandibular-derived primary culture is much more crowded and contains polygonal cells.
  • the articular-derived culture is very sparse. Most of the cells do not adhere to the plate.
  • the non-adherent cells form refractive-shiny aggregates of cells, while the adherent cells are elongated resembling fibroblast like cells.
  • Four days following the first culture split the mandibular cells are already organized in typical culture nodule, while most the articular cells remain elongated.
  • FIG. 3A shows the levels of thymidine incorporation into DNA in the chondrocytes of the two sources. Normally proliferation rate declines in the developing culture as the cells undergo differentiation. Yet, at each time interval tested: 24, 48 and 72 hr, the levels of thymidine incorporation into the AC-derived chondrocytes was lower (by 70%, 30% and 13% respectively) than that of the MC-derived chondrocytes. The lower proliferation activity is reflected eventually in fewer AC-derived viable cells compared to the MC ones as apparent from the lower MTT activity ( figure 3B ).
  • Normal joint surfacing cartilage is hyaline cartilage, which is characterized by type II collagen and aggrecan-rich matrix.
  • type II collagen determined using immunohistochemical (IHC) analyses ( figure 4B ) clearly show intensive positive staining at both 4 (panels A and B) and 17 days (panels C and D) MC culture and a very faint (mainly background) positive staining in the AC-derived chondrocytes.
  • Densitometry of the type II collagen staining ( figure 4A ) reveals a 24%, 44% and 73% (after 4, 10 and 17 days, respectively) decrease in the AC positive type II collagen staining compared to that of the MC.
  • Aggrecan is a specific cartilaginous proteoglycan rich in sulfated glycosaminoglycans and serves as one of the major components of hyaline cartilage.
  • Immunohistochemical analysis of 4 and 17 day old cultures ( figure 5A ) reveal markedly higher positive aggrecan activity in MC chondrocytes as compared to the AC-derived chondrocytes.
  • Maturation and senescence of cartilage is typified by a decrease in matrix production reflected in decreasing rates of proteoglycan sulphation.
  • Newly produced aggrecan may be determined by following incorporation of radiolabeled sulphate into CPC (cetyl pyridinium chloride) insoluble proteoglycan fraction.
  • CPC cetyl pyridinium chloride
  • Incorporation of sulphate into proteoglycans was assayed as described above. Results shown in figure 5C show a 37% decrease in sulphate uptake by MC between 9 and 14 days.
  • hyaline cartilage In addition to the expression of cartilaginous specific genes, normally differentiating hyaline cartilage is also characterized by its concomitantly decreasing production of type I collagen.
  • the expression of type I collagen was assayed immunohistochemically. Densitometry of IHC staining ( Figure 6A ), show that while the expression of type I collagen is gradually reduced in the MC-derived chondrocytes, the expression of type I collagen by AC cells gradually increases and reaches 79%, 146% and more than 140% higher levels than those of MC-derived cell, after 4, 10 and 17 days respectively indicating formation of fibrocartilage.
  • Type I collagen is associated with fibrocartilage, which is relatively inferior in terms of strength and durability compared to hyaline cartilage.
  • Porcine mandibular condyle-derived chondrocytes from the second passage were plated on cover glasses at a concentration of 5 ⁇ 10 5 cells/ml in 35 mm culture wells and cultured under same conditions as described above for MCDC. Cells were re-fed every 48 hr. At 12 days post-plating, an intact cartilage film developed. The cartilage film was rigid enough to be transferred and re-plated in a new culture dish. Throughout the first 24 hr, cartilage film was cultured with only 1 ml of medium to let it adhere to the plate. Further culturing continued under ordinary conditions. Development of the cartilage culture originating from the original cartilage film was followed morphologically and biochemically.
  • Mandibular condyle-derived chondrocytes rapidly differentiate in culture into cartilage forming cells, which secrete increasing amounts of type II collagen and aggrecan.
  • an initial cartilaginous film is formed.
  • This cartilage tissue gradually thickens and by about the twelfth day becomes a solid membrane, which can be mechanically manipulated while preserving its original features to proliferate and re-differentiate.
  • the cartilaginous film was replated in culture medium to test its ability to proliferate following transfer. A cartilaginous film that undergoes hyperproliferation upon transfer to new growth medium would be highly undesirable for cartilage repair.
  • Figure 7 presents photos taken 4, 8 and 14 days post re-plating of the formed 12-day old cartilage film.
  • New cells (NC) began to sprout out from the 'old' cartilage film (CF). After 8 days these new chondrocytes began to develop polygonal shapes ( figure 7B ).
  • two weeks post re-plating a new typical chondrocyte culture developed ( figure 7C ) occupying most of the new culture well. However the new culture did not exhibit cellular over-growth, and is clearly restrained by a border of cells ( figure 7D , arrow), indicating that the secondary new culture (NC) preserves features of a primary culture and does not exhibit hyperproliferation.
  • NC secondary new culture
  • Cartilage film preserves its capacity to undergo neo-proliferation upon replating.
  • the cartilage film exhibits the essential features for cartilage repair: the ability to proliferate and form hyaline cartilage in situ .
  • AIA Adjuvant induced rheumatoid arthritis
  • Rats were kept separately to recover from transplantation. About three-four weeks after transplantation most of the treated rats moved more freely compared to the positive control. After 60 days, rats were sacrificed and knee joints were excised and processed for histological analysis.
  • Figure 9A shows the articular cartilage of a control animal (C-); figure 9B shows a cartilage cross section from a positive control animal in which AIA was induced; allogenic transplantation of rat MCDC cells transplanted into rat (9C), xenogeneic transplantation of mouse MCDC cells transplanted into rat (9D) and xenogeneic transplantation of porcine MCDC cells transplanted into rat (9E).
  • C- control animal
  • Figure 9B shows a cartilage cross section from a positive control animal in which AIA was induced; allogenic transplantation of rat MCDC cells transplanted into rat (9C), xenogeneic transplantation of mouse MCDC cells transplanted into rat (9D) and xenogeneic transplantation of porcine MCDC cells transplanted into rat (9E).
  • Example 4 Implantation of chondrocyte film into goats
  • Goats were operated on their right leg generating two 10mm diameter by 2 mm deep lesions, one in the medial femoral condyle and the second in the lateral femoral condyle.
  • the medial lesion was covered by a chondrocyte film prepared from porcine MCDC, and sutured to the lesion margin; while the lateral lesion was left untreated.
  • a 12 day old cartilage film 25 mm diameter by 0.1 mm wide was implanted into the formed lesion; the surgical opening was closed. Following 10 days of indoor housing, the goats returned to the farm and were given unrestricted activity.
  • the articular surface of the knee joint Upon euthanasia, the articular surface of the knee joint will be examined by 3 blinded observers and scored for surface smoothness, integration of the lesion-fill edge and transparency of the filled tissue.
  • synovial membrane and joint capsule were observed and photographed for morphological changes and particularly any inflammation signs.
  • the nearest lymphatic node will be examined and processed for histological analyses.
  • Synovial fluid was collected for analyzing cells population and immunoglobulin proteins content. Parallel analyses are conducted on the intact /counterpart leg.
  • Synovial fluid composition was similar in the operated (right) and intact (left) joint. Although the operated joint contained more fluid, the total neutrophil cell number was identical. Goats move freely, eat and behave normally. All blood test results were within normal ranges; including white blood cell (WBC) count. Joint gross morphology was unchanged with no signs of edema. Synovial fluid drained 3 months after implantation had no signs of inflammation.
  • WBC white blood cell
  • Results shown in table 1 clearly show that the increased SF volume in the treated joint is not due to inflammatory reaction, but rather to normal healing process often followed by increased secretion of SF.
  • the total neutrophil number indicating a potential inflammatory state, is similar in both joints; (in the left joint the neutrophil concentration is 4 times higher, in a 4 times smaller volume).
  • Table 1 Synovial Fluid Joint Total fluid WBC Neutrophils Right leg (operated) 1000 ⁇ l 0.2 x 10 3 / ⁇ l 4% Left (intact) 250 ⁇ l 0.24 x 10 3 / ⁇ l 19.6%
  • Lesion dimensions are shown in table 2.
  • the untreated lesion size (on the lateral condyle) increased by 21% while the treated lesion size (on the medial condyle) decreased by 20%.
  • Table 2 Changes in lesions size after 3 months. Joint Original lesion size Lesion size after 14 weeks Right 314 mm 2 254 mm 2 Left 314 mm 2 380 mm 2
  • Photographs of the lesions are presented in Figure 10 .
  • the treated-medial lesion is rounded and shows signs of initial cartilage covering at the lesion's margins ( Figure 10C ).
  • the lateral lesion (untreated) has developed an irregular shape, indicating a possible preliminary secondary osteoarthritic process, a well known phenomenon that often accompanies untreated articular lesions ( Figure 10B ).
  • the implanted chondrocytes are derived from male porcine and the recipient is female goat.
  • a Y-chromosome specific gene will serve for localizing the porcine-derived cells within the recovered articular lesion and to trace any porcine originated cells that might leak out of the implant into the synovial fluid.
  • the expression of the porcine Y gene will be localized in the synovial fluid and in osteochondral biopsies' sections of the using PCR and in situ hybridization respectively.
  • Biomechanical evaluations of the newly formed cartilage, necropsies of osteochondral (articular cartilage and its subchondral bone) harvested from the treated lesions, the untreated lesions and from parallel healthy tissues in the counter (intact) knee, will be made immediate post tissue excising.
  • Specimens are immersed in Phosphate-Buffered Saline (PBS, pH 7.2) containing protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 2 mM disodium ethylenediamine tetraacetate, 5 mM benzamidine-HCl, and 10 mM N-ethylmaleimide). Tissues are assayed within 30 min of their harvesting.
  • PBS Phosphate-Buffered Saline
  • Rigidity of the tissue is assessed by indentation test using 'universal testing apparatus 4502'-Instron-BioplusTM, and a 0.3mm plane ended indenter.
  • the loading protocol consists of application of a "tare displacement” (for 30 s) and then four "test displacements" of 3 s duration each. For each tare displacement-load at 0 s and 3 s is noted. Tare displacement is 25 micrometer, test displacements are 50,100, 200, and 300 micrometers.
  • Indentation Stiffness is computerized calculated as the slope of load-displacement data, in units of N/mm (with no need to regard indenter diameter).
  • explants will be cut at a perpendicular axis to the lesion, through the center: one section will be used for histological/histochemical studies and the other for biochemical analyses.
  • Explants are fixed with NBF (neutral buffered formalin), decalcified for 48 hr in EDTA, dehydrated in gradual alcohols and routinely processed for paraffin embedding.
  • NBF neutral buffered formalin
  • the second explant samples will serve for extraction of lysate proteins, DNA and RNA for immunoblotting, PCR and real time PCR analysis respectively.
  • RT-PCR Real time PCR
  • ISH In situ hybridization
  • Dig-labeled probes are produced by two PCR steps. The first product will be produced using primers detailed below. The first PCR product is subjected to PCR amplification using promoter-primer sequence combined to the gene specific primer as brought in the table. The second product serves as a template for either sense (negative control) or antisense (in situ hybridization) Dig-labeled RNA using (Sp6/T7) Dig-RNA labeling kit (Roche, Germany), following the manufacturer's instructions. Hybridization conditions and detection will be performed as described below.
  • a Dig labeled DNA probe will be performed, using PCR Dig probe synthesis kit (Roche).
  • Table 3 provides exemplary primer sequences for use in, inter alia , the RT-PCR and ISH procedures.
  • Table 3 Primer sequence size Collagen type II - sense T7 promoter + TCTCCTGCCTCCT (SEQ ID NO :1) 302bp Collagen type II - antisense T3 promoter + CCATCTCTGCCACGG (SEQ ID NO :2) Aggrecan forward T7 promoter + GTCCTCTCCAGT (SEQ ID NO :3) 345bp Aggrecan reverse T3 promoter + TTGCTTCTCCAGA (SEQ ID NO :4) Porcine Y-chromosome -sense T7+AAGTGGTCAGCGTGTCCATA (SEQ ID NO :5) 236 bp Porcine Y-chromosome antisense T3 + TTTCTCCTGTATCCTCCTGC (SEQ ID NO :6) Collagen type X - antisense T7 promoter + GATCCTCACATA (SEQ ID NO :7) 481bp Collagen type
  • the T7 promoter primer sequence is 5' CAAGCTTCTAATACGACTCACTATAGGGAGA 3' (SEQ ID NO:11);
  • the T3 promoter primer sequence is 5' CCAAGCTTCATTAACCCTCACTAAAGGGAGA 3'(SEQ ID NO:12).
  • ISH In situ hybridization
  • In situ hybridization is performed on deparaffinized sections loaded on precleaned poly-L-lysine-coated slides. Sections are treated with 3% H2O2 in methanol to neutralize endogenous peroxidase. After permeabilization with 12.5 mg/ml proteinase K for 15 min, (stopped with 2 mg/ml glycine), and acetylation in 0.5% acetic anhydride in 0.1 mol/L Tris at pH 8.0, sections are post-fixed with 4% paraformaldehyde/phosphate buffered saline (PBS).
  • PBS paraformaldehyde/phosphate buffered saline
  • Hybridization for 10 min in 2 x standard saline citrate (2xSSC) is followed by one hour in hybridization buffer: 50% formamide, 0.5 mg/mL salmon sperm DNA, 4xSSC, 1x Denhardt's, 200 U/ml heparin, 5% dextran sulfate and 0.01% sodium dodecyl sulfate (SDS).
  • Hybridization is carried out for 18hr at 42°C and maximal humidity with a 5mg/mL digoxygenin labeled antisense RNA probe (see above). Digoxygenin labeled sense RNA probes were run as negative controls.
  • slides are rinsed in SSC under increasingly stringent conditions and then with 0.1mol/L Tris, 0.15 mol/L NaCl at pH 7.5.
  • Hybrids are detected using anti-digoxygenin antibodies conjugated with peroxidase (Boehringer Mannheim, Germany) and AEC as a substrate; counterstaining is done with hematoxylin.
  • slides will undergo similar pre-treatment except for acetylation. Prior to hybridization, slides- covered with the labeled probe cocktail, are prewarmed for 6 min at 95°C, cool immediately and placed to hybridize for 3 hrs at 42°C.
  • Mandibular condyle derived chondrocytes provide a unique source of cartilage producing cells different from other cartilage sources, in particular from articular cartilage (AC).
  • AC is the standard source for chondrocytes used in replacement procedures and the only one currently approved for autologous cartilage transplantation (ACT).
  • MC is preferable to AC as a source for cartilage producing cells for the purpose of treating articular lesions from both efficacy and competency aspects. Viable cell population yielded from MC is much higher than that of AC. The cells are more metabolically active and proliferate at a higher rate than AC chondrocytes. Most important, MC derived chondrocytes express higher levels of type II collagen and aggrecans than AC cells, indicating spontaneous differentiation of MC cells into hyaline cartilage producing cells. AC cultures, in contrast, produce decreasing amounts of type II collagen and aggrecan with culturing time, and express increasing amounts of type I collagen, indicating development of potentially detrimental fibrocartilage.
  • Prolonged MCDC cultures develop into cartilage film composed of chondrocytes and extracellular matrix containing mainly type II collagen and aggrecans. After 10-14 days of culture the cartilage film is rigid enough to be mechanically manipulated yet pliable enough to adapt to various contours of particular cartilage's shape. It can be transferred from one dish to another as an intact tissue (replating). Upon transfer, the recultured film regains developmental activities featuring the newly cultured MCDC chondrocytes, indicating that the cartilage film preserves de novo chondrogenesis potential including cell proliferation and differentiation. At the margins of the film, cells start to proliferate, followed by differentiation into hyaline cartilage. The growth of the cartilage is restrained, and no overgrowth of cartilage tissue is observed. These unique features of the cartilage film provide a product suitable for cartilage replacement since it guarantees in situ development of vital, high quality hyaline cartilage.
  • RA rheumatoid arthritis
  • AIA Adjuvant Induced rheumatoid Arthritis
  • Newly formed cartilage displayed typical developmental cell gradient restrained to the anatomical boundaries of the apical epiphysis. Moreover, no signs of any immunological response were noticed and WBC were not observed.

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